James W. B. Moir

Respiratory detoxification of nitric oxide by the cytochrome c nitrite reductase of Escherichia coli

Nitric oxide is a key element in host defense against invasive pathogens. The periplasmic cytochrome cnitrite reductase (NrfA) of Escherichia coli catalyzes the respiratory reduction of nitrite, but in vitro studies have shown that it can also reduce nitric oxide. The physiological significance of the latter reaction in vivo has never been assessed. In this study the reduction of nitric oxide byEscherichia coli was measured in strains active or deficient in periplasmic nitrite reduction. Nrf+ cells, harvested from cultures grown anaerobically, possessed a nitric-oxide reductase activity with physiological electron donation of 60 nmol min−1· mg dry wt−1, and an in vivo turnover number of NrfA of 390 NO⋅ s−1was calculated. Nitric-oxide reductase activity could not be detected in Nrf− strains. Comparison of the anaerobic growth of Nrf+ and Nrf− strains revealed a higher sensitivity to nitric oxide in the NrfA− strains. A higher sensitivity to the nitrosating agentS-nitroso-N-acetyl penicillamine (SNAP) was also observed in agar plate disk-diffusion assays. Oxygen respiration by E. coli was also more sensitive to nitric oxide in the Nrf− strains compared with the Nrf+ parent strain. The results demonstrate that active periplasmic cytochromec nitrite reductase can confer the capacity for nitric oxide reduction and detoxification on E. coli. Genomic analysis of many pathogenic enteric bacteria reveals the presence ofnrf genes. The present study raises the possibility that this reflects an important role for the cytochrome cnitrite reductase in nitric oxide management in oxygen-limited environments.

Nitrate and nitrite transport in bacteria

Abstract. The topological arrangements of nitrate and nitrite reductases in bacteria necessitate the synthesis of transporter proteins that carry the nitrogen oxyanions across the cytoplasmic membrane. For assimilation of nitrate (and nitrite) there are two types of uptake system known: ABC transporters that are driven by ATP hydrolysis, and secondary transporters reliant on a proton motive force. Proteins homologous to the latter type of transporter are also involved in nitrate and nitrite transport in dissimilatory processes such as denitrification. These proteins belong to the NarK family, which is a branch of the Major Facilitator Superfamily. The mechanism and substrate specificity of transport via these proteins is unknown, but is discussed in the light of sequence analysis of members of the NarK family. A hypothesis for nitrate and nitrite transport is proposed based on the finding that there are two distinct types of NarK.

Nitric oxide in bacteria: synthesis and consumption

5. Nitric oxide signalling in bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466 5.1. Denitrifying bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466 5.2. Pathogenic bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467 5.3. Parallels with eukaryotic systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468

Nitric Oxide Detoxification Systems Enhance Survival of Neisseria meningitidis in Human Macrophages and in Nasopharyngeal Mucosa

ABSTRACT Nitric oxide (NO) contributes to mammalian host defense by direct microbicidal activity and as a signaling molecule of innate immune responses. Macrophages produce NO via the inducible NO synthase (iNOS). The genome of Neisseria meningitidis includes two genes, norB (encoding nitric oxide reductase) and cycP (encoding cytochrome c′), both of which detoxify NO in pure cultures of N. meningitidis. We show here that norB, and to a lesser extent cycP, enhance survival of N. meningitidis within primary human macrophages. Furthermore, accumulation of lysosome-associated membrane protein 1 (LAMP-1) is modified in phagosomes containing an isogenic norB mutant of N. meningitidis compared to the wild type. The survival enhancement conferred by norB and cycP is ablated by pretreatment of macrophages with the nitric oxide synthase inhibitor N(G)-monomethyl-l-arginine (L-NMMA). Despite this evidence that NO detoxification confers advantage, we find, using a highly sensitive chemiluminescence technique, that human macrophage-associated [NO] is low even after activation by lipopolysaccharide and interferon alpha. Furthermore, wild-type N. meningitidis further depletes cell-associated NO during phagocytosis by an active mechanism and survives relatively poorly in the presence of L-NMMA, suggesting that the wild-type organism may utilize NO for optimal survival during intracellular life. The natural habitat of N. meningitidis is the human nasopharynx. Using a nasopharyngeal mucosa organ culture system, we show that mutants lacking norB and cycP also survive poorly in nasopharyngeal tissue compared to wild-type N. meningitidis. These findings indicate that the meningococcus requires active NO detoxification systems for optimal survival during experimental nasopharyngeal colonization and processing by human phagocytic cells.

The purification of ammonia monooxygenase from Paracoccus denitrficans

The heterotrophic nitrifier Paracoccus denitrificans expresses a membrane‐associated ammonia monooxygenase. The active enzyme has been solubilized in the detergent dodecyl‐β‐d‐maltoside and purified by standard chromatographic techniques. This is the first purification of an ammonia monooxygenase. The enzyme consists of two subunits with molecular masses of 38 and 46 kDa. The purified enzyme is a quinol oxidase, is inhibited by light and a variety of chelating agents and is activated by cupric ions. These properties indicate that this enzyme has similarities to a family of enzymes including the ammonia monooxygenase from Nitrosomonas europaea and the particulate methane monooxygenase from Methylococcus capsulatus (Bath).

Two domains of a dual‐function NarK protein are required for nitrate uptake, the first step of denitrification in Paracoccus pantotrophus

Uptake of nitrate into the cytoplasm is the first but least well understood step of denitrification; no gene has previously been identified to be necessary for this process. Upstream from the structural genes of the membrane‐bound nitrate reductase (narGHJI) in Paracoccus pantotrophus there is a fusion of two genes, each homologous to members of the narK family. The single open reading frame is predicted to encode 24 transmembrane helices, comprising two domains, NarK1 and NarK2. Analysis of both the accumulation of intracellular nitrite and electron transport through the nitrate reductase enzyme in narK mutants reveals that NarK1 and NarK2 are both involved in nitrate uptake. Maximal rate of nitrate transport via NarK2 was dependent upon nitrite, indicating that NarK2 encodes a nitrate/nitrite antiporter. The uncouplers S13 and dinitrophenol showed that NarK2 was not dependent on the proton motive force for maximal nitrate transport activity. Nitrate transport via NarK1 was dependent on proton motive force, indicating that it is likely to be a nitrate/proton symporter. Low expression of membrane‐bound nitrate reductase in narK mutants was counteracted by azide, which induced nitrate reductase expression only if the transcriptional activator NarR was present.

Enhanced glycosylation with mutants of endohexosaminidase A (endo A).

Glycosylation of proteins is the most diverse form of posttranslational modification, and can play a key role in protein folding, and can also crucially affect important protein properties. However, since the biosynthesis of glycans is not under direct genetic control, glycoproteins are produced intracellularly as heterogeneous mixtures of glycoforms, in which different oligosaccharide structures are linked to the same peptide chain. Access to pure single glycoforms of glycoproteins has now become a major scientific objective since it is not only a prerequisite for more precise biological investigations into the different effects glycans have on protein properties, but also an important commercial goal in the field of glycoprotein therapeutics, which are currently marketed as heterogeneous mixtures of glycoforms. Access to single glycoforms of glycoproteins can be achieved by total synthesis of both glycan and polypeptide components, and some outstanding achievements in this area have recently been published. However, such ACHTUNGTRENNUNGsynthesis approaches are particularly arduous and do not realistically represent a practical approach that could be applied to widespread and large-scale glycoprotein production. Alternative approaches based on bioengineering of cell lines in order to optimise production of glycoproteins that bear particular oligosaccharide structures have also been reported and are being exploited commercially, though such approaches have no guarantee of complete glycan homogeneity. An alternative method for achieving homogeneous protein glycosylation involves the use of enzymatic catalysis, and one particular class of enzyme that displays considerable synthesis potential in this respect comprises the endohexosaminidases. Endohexosaminidases are a class of enzyme that specifically cleave the chitobiose core [GlcNAcb ACHTUNGTRENNUNG(1-4)GlcNAc] of Nlinked glycans between the two N-acetyl glucosamine residues, and since they cleave this linkage they can also be used to selectively synthesise it. Two members of this class that have been demonstrated to display useful synthesis glycosylation activity are Endo M from Mucor hiemalis and Endo A from Arthrobacter protophormiae. However, since these enzymecatalysed reactions are reversible, competitive product hydrolysis can greatly reduce synthesis efficiency, particularly when transglycosylations are undertaken with unactivated donors. Seminal work in the field by Shoda and co-workers demonstrated that carbohydrate oxazolines are useful activated glycosyl donors for these enzymes, presumably because they mimic the putative oxazolinium ions, which are proposed intermediates in the enzyme-catalysed hydrolysis reaction. Subsequently, extensive work from the group of Wang has detailed the efficient synthesis of a series of glycopeptides by transglycosylation with Endo A; they have also recently reported the synthesis of single glycoforms of ribonuclease B by using this approach. In order to circumvent the problem of competitive product hydrolysis previous work has focussed on the attempted development of irreversible glycosylation reactions with structurally modified oxazolines as glycosyl donors; the synthesis products of these reactions are generally not hydrolysed by the endohexosaminidase used to promote their synthesis ; the enzymes therefore act as glycoligases. However, another potential way to circumvent this problem is to either use specifically mutated enzymes or glycosynthases—as developed by Withers and Planas—which are not capable of product hydrolysis. The term “glycosynthase” was first applied by Withers to retaining glycosidases in which the nucleophilic of the two catalytic acid residues in the enzyme active site had been replaced by site-directed mutagenesis with a nonparticipating residue, for example, by alanine. The use of an activated glycosyl donor, such as a glycosyl fluoride, allows this mutant enzyme to promote a synthesis reaction, but the mutant enzyme is not capable of hydrolysing the product glycosidic linkage as the key nucleophilic residue was absent. Endo A is a member of family 85 of the glycohydrolases (GH85). These enzymes, though they are retaining glycosidases, are thought to catalyse hydrolysis by a neighbouringgroup-participation mechanism in which the carbonyl oxygen of the 2-acetamide of the second GlcNAc residue is the actual nucleophile, rather than an enzyme-bound aspartate or glutamate. These enzymes, therefore, do not possess a nucleophilic residue at the active site, and as such it is not possible to envisage the production of a glycosynthase along the lines of the accepted Withers and Planas precedents. However, Wang et al. have very recently reported the production of a series of mutants of Endo M—another family 85 endohexosaminidase— which they screened for hydrolytic and transglycosylation activity. In particular they identified an N175A mutant of Endo M in which Asn175—a conserved residue in the GH85 family— was replaced by alanine; this mutant displayed glycosynthase activity by using oxazolines as glycosyl donors. Since this N175A mutant displayed only marginal hydrolysis activity it [a] Dr. C. D. Heidecke, T. B. Parsons, Dr. A. J. Fairbanks Department of Chemistry, Chemistry Research Laboratory University of Oxford, Mansfield Road, Oxford, OX1 3TA (UK) Fax: (+44)1865-275674 E-mail : antony.fairbanks@chem.ox.ac.uk [b] Dr. Z. Ling, Prof. N. C. Bruce, Dr. J. W. B. Moir Department of Biology, University of York YO10 5YW (UK) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

Regulation of Denitrification Genes in Neisseria meningitidis by Nitric Oxide and the Repressor NsrR

The human pathogen Neisseria meningitidis is capable of growth using the denitrification of nitrite to nitrous oxide under microaerobic conditions. This process is catalyzed by two reductases: nitrite reductase (encoded by aniA) and nitric oxide (NO) reductase (encoded by norB). Here, we show that in N. meningitidis MC58 norB is regulated by nitric oxide via the product of gene NMB0437 which encodes NsrR. NsrR is a repressor in the absence of NO, but norB expression is derepressed by NO in an NsrR-dependent manner. nsrR-deficient mutants grow by denitrification more rapidly than wild-type N. meningitidis, and this is coincident with the upregulation of both NO reductase and nitrite reductase even under aerobic conditions in the absence of nitrite or NO. The NsrR-dependent repression of aniA (unlike that of norB) is not lifted in the presence of NO. The role of NsrR in the control of expression of aniA is linked to the function of the anaerobic activator protein FNR: analysis of nsrR and fnr single and nsrR fnr double mutants carrying an aniA promoter lacZ fusion indicates that the role of NsrR is to prevent FNR-dependent aniA expression under aerobic conditions, indicating that FNR in N. meningitidis retains considerable activity aerobically.

Endohexosaminidase M : Exploring and exploiting enzyme substrate specificity

Post-translational modification of proteins by glycosylation can play a key role in protein folding and can also crucially affect important protein properties such as conformation and stability, susceptibility to proteases and circulatory lifetime. In addition, protein glycosylation plays a role in many other key biological processes such as cell–cell signalling, development and immune response. The synthesis of the carbohydrate portion of glycoproteins is not under direct genetic control, and, as a result, glycoproteins are typically biosynthesised as complex heterogeneous mixtures, known as glycoforms, in which different oligosaccharide structures are linked to the same peptide chain. These mixtures are to all intents and purposes inseparable, since their physical properties are so similar. However, access to pure single glycoforms of glycoproteins is not only a prerequisite for precise biological investigation but is also becoming an increasingly important goal in relation to glycoprotein therapeutics, which are currently marketed as heterogeneous glycoform mixtures. Whilst several chemoselective methods have been developed in order to synthetically access proteins glycosylated with defined oligosaccharides, 10] these methods suffer from the disadvantage that the carbohydrates are connected to the peptide backbone by non-native linkages. As an alternative method for achieving homogenous protein glycosylation, several groups have recently promoted the use of enzyme catalysis. One particular class of enzymes that display considerable synthetic potential in this respect is the endohexosaminidases, which specifically cleave the chitobiose core [GlcNAcbACHTUNGTRENNUNG(1– 4) lcNAc] of N-linked glycans between the two N-acetyl glucosamine residues. Two members of this class that have been shown to display useful synthetic glycosylation activity are Endo M from Mucor hiemalis and Endo A from Arthrobacter protophormiae. Previous work, particularly on the use of Endo A, has demonstrated that carbohydrate oxazolines are useful activated glycosyl donors for these enzymes, presumably since they mimic the putative oxazolonium ions, which are intermediates in the enzyme-catalysed hydrolysis reaction. Indeed, the efficient synthesis of a series of glycopeptides has been achieved by transglycosylation with Endo A; moreover a recent paper also details investigations of a correlation between the efficiency of glycosylation and substrate structure. However, as with many enzyme-catalysed transglycosylations, one particular problem that can greatly reduce synthetic efficiency and utility is product hydrolysis, since, in general, the product is itself an enzyme substrate. One particularly elegant way of circumventing this problem is the use of specifically mutated enzymes, or so called glycosynthases, developed by Withers and Planas, which are not capable of product hydrolysis. Another potential solution to this problem is the use of highly activated donors that are structurally slightly modified. In this case, the activated donor could well be processed by the enzyme at a reasonably efficient rate, particularly if the donor itself can be considered as a transition-state mimic, and yet the product might not be hydrolysed due to the minor structural modification. As part of a long-term program aimed at developing synthetic routes to pure, single glycoforms of glycoproteins, we recently became interested in the use of Endo M as a catalyst for the conjugation of synthetic oligosaccharides to glycopeptides and proteins bearing single GlcNAc residues. Herein we report investigations into the substrate tolerance and efficiency of glycosylation for Endo M-mediated reactions of a model glycosyl amino acid acceptor with a range of natural N-glycan oxazoline donors, and also several donors that contain a minor structural modification. Oxazoline donors 1–4 (Scheme 1), which correspond to fragments of natural high-mannose N-glycans, were synthetically accessed as previously described. In addition, in order to probe the substrate tolerance of Endo M and to be able to later investigate the importance and role of the central mannose unit of the core N-glycan pentasaccharide, the gluco-containing diand trisaccharide oxazolines 5 and 6 were also ACHTUNGTRENNUNGaccessed as follows. Methyl triflate-mediated glycosylation of the gluco thioglycoside donor 7 with protected glucosamine acceptor 8 gave the bACHTUNGTRENNUNG(1–4)-linked disaccharide 9, which was then converted into the deprotected oxazoline 5 by following a series of standard protecting-group manipulations and finally Zemplen deacetylation (Scheme 2). Likewise, the previously described ACHTUNGTRENNUNGglucose-containing trisaccharide 13 was converted into the ACHTUNGTRENNUNGdeprotected trisaccharide oxazoline 6 by following a similar series of protecting-group manipulations, and a final global ACHTUNGTRENNUNGdeprotection step, again by Zemplen deacetylation. Endo M-catalysed glycosylations were carried out by using model glycosyl amino acid 17 as the acceptor, since it possessed Z protection of nitrogen as a chromophore to facilitate HPLC analysis (Scheme 3). Endo M-catalysed reaction of monosaccharide donor 1 yielded no product; this indicated that at least a disaccharide is required as the minimum donor structure. That only a minimum of disaccharide is required for efficient glycosylation was confirmed by observing that the ManGlcNAc disaccharide oxazoline donor 2 glycosylated acceptor 17 to give trisaccharide 19 in a yield of 98% after 3 h [a] T. W. D. F. Rising, Dr. T. D. W. Claridge, Dr. A. J. Fairbanks Chemistry Research Laboratory, Oxford University Mansfield Road, Oxford, OX1 3TA (UK) Fax: (+44)1865-275674 E-mail : antony.fairbanks@chem.ox.ac.uk [b] Dr. J. W. B. Moir Department of Biology, University of York York, YO10 5YW (UK) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

The pathogen Neisseria meningitidis requires oxygen, but supplements growth by denitrification. Nitrite, nitric oxide and oxygen control respiratory flux at genetic and metabolic levels

The human pathogen Neisseria meningitidis is the major causative agent of bacterial meningitis. The organism is usually treated as a strict aerobe and is cultured under fully aerobic conditions in the laboratory. We demonstrate here that although N. meningitidis fails to grow under strictly anaerobic conditions, under oxygen limitation the bacterium expresses a denitrification pathway (reduction of nitrite to nitrous oxide via nitric oxide) and that this pathway supplements growth. The expression of the gene aniA, which encodes nitrite reductase, is regulated by oxygen depletion and nitrite availability via transcriptional regulator FNR and two‐component sensor‐regulator NarQ/NarP respectively. Completion of the two‐step denitrification pathway requires nitric oxide (NO) reduction, which proceeds after NO has accumulated during batch growth under oxygen‐limited conditions. During periods of NO accumulation both nitrite and NO reduction are observed aerobically, indicating N. meningitidis can act as an aerobic denitrifier. However, under steady‐state conditions in which NO is maintained at a low concentration, oxygen respiration is favoured over denitrification. NO inhibits oxidase activity in N. meningitidis with an apparent Ki NO = 380 nM measured in intact cells. The high respiratory flux to nitrite after microaerobic growth and the finding that accumulation of the denitrification intermediate NO inhibits oxygen respiration support the view that denitrification is a pathway of major importance in N. meningitidis.